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Studies on ADP-Ribose Polymer Metabolism in Cultured Mammalian Cells Following DNA Damage

Description: ADP-ribose polymer metabolism has been studied in human cells derived from a patient with Glutamyl Ribose Phosphate Storage Disease (GRPSD) and in mouse C3H1OT1/2 cells following oxidative stress induced by hydrogen peroxide (H202 ). It has been postulated that GRPSD resulted from an abnormality in ADP-ribose polymer metabolism. This study has shown that these cells exhibit reduced poly(ADP ribose) polymerase activity which is proposed to result from modification of the enzyme with ribose phosphate groups. The modification in the polymerase is proposed to be secondary to a defect in either ADP-ribosyl proteinlyase or an overproduction of a cellular phosphodiesterase. The metabolism of ADP-ribose polymers was rapidly altered by H202 and there were independent effects on adenine nucleotide pools. The results suggest that ADP-ribose polymer metabolism is involved in cellular defenses to oxidative stress.
Date: May 1991
Creator: Maharaj, Geeta
Partner: UNT Libraries

Opthalmic Use Of Sodium Cephalothin: An In Vivo Comparison

Description: A rabbit keratoconjunctivities model was used to evaluate ophthalmic formulations containing 1 percent sodium cephalothin in silicon oil, a 1 percent sodium cephalothin aqueous solution, and a 0.3 percent gentamicin sulfate solution. Rabit eyes were inoculated intracorneally with Pseudomonas aeruginosa, Staphylococcus aureus, or Streptococcus pneumoniae, After topical treatment, none of the antibiotic formulations were effective in the P. aeruginosa model; all three showed good activity against S. aureus, and against S. pneumoniae, the caphalothin formulations were more effective than gentamicin.In a related stability study, the cephalothin potency of the silicon formulation was maintained for 16 weeks at 4, 25, and 450 C These studies suggest that sodium cephalothin can be formulated as an effective and stable ophthalmic dosage form.
Date: August 1979
Creator: Carney, Gerald R.
Partner: UNT Libraries

Regulation of Pyridine Nucleotide Metabolism in Saccharomyces cerevisiae

Description: The levels of total nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), and their redox states were determined as the function of growth in S. cerevisiae. Cells growing in a medium containing 0.8% glucose exhibit two phases of exponential growth, utilizing glucose and ethanol, respectively. The NAD pool is 50% reduced during both stages of growth while the NADP pool is 67% reduced in glucose growth and 48% reduced in ethanol growth. The NAD/NADP ratio is constant during growth on glucose and a two-fold increase in the NAD/NADP ratio occurs upon exhaustion of glucose. The increased ratio is maintained during growth on ethanol. This alteration in the regulation of the relative levels of NAD and NADP may be due to a change in the regulation of NAD kinase and/or NADP phosphatase activities. These changes may be related to the redox state of the NADP pool.
Date: May 1976
Creator: Ting, Haung-yu
Partner: UNT Libraries

Chemical Cleavage of Human Phosphoglucose Isomerase at Cysteine

Description: The present study has resulted in the development of a procedure for the specific chemical fragmentation of human phosphoglucose isomerase into a minimal number of peptides. A two-cycle procedure for cleaving the protein with 2-nitro-5- thiocyanobenzoic acid results in four primary peptides and three overlap peptides. The peptides can be readily separated on the basis of their size by using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Preliminary peptide alignments have been considered, and amino acid analyses have been performed. End-terminal analyses of the enzyme revealed a carboxyl terminal sequence of Asp-Val-Gln and a blocked amino terminus. The cysteine cleavage procedure provides an excellent method for the identification and location of specific genetic mutations of human phosphoglucose isomerase.
Date: December 1975
Creator: Conn, Worth R.
Partner: UNT Libraries

The Photolytic Ozonation of Organics in Aqueous Solutions

Description: The aim of the investigation described in this work is to gain a better understanding of the processes involved in the oxidation of organic compounds by photolytic ozonation in a laboratory scale reactor. The results and discussions are presented in Chapter III. This chapter contains four parts. In the first part, mass transfer efficiency and the calculation of the mass transfer coefficient, KLa, as well as the ozone decomposition rate constant, KD, are presented and compared with those obtained by other investigators. The second part deals with the kinetics of the photolysis of 2,2',4,4',6,6'-hexachlorobiphenyl both in purified and natural lake water. Mathematical expressions and a discussion of the possible reaction processes involved are given. Kinetic models of ozonation and photolytic ozonation in purified and natural lake water are developed and discussed in part three. Rate constants are calculated from experimental data and used to predict values of substrate destruction with a mathematical model. The fourth part of this chapter deals with the identification of products from the photolysis, ozonation and photolytic ozonation of 2,2'4,4',6,6'-hexachlorobiphenyl. The products are isolated and identified using combined gas chromatography and mass spectroscopy, and reaction mechanisms are suggested.
Date: May 1980
Creator: Huang, Francis Y.
Partner: UNT Libraries

Modulation of ³H-Myo-Inositol Uptake by Glucose and Sorbitol in Cultured Bovine Lens Epithelial Cells

Description: Myo-[3H]-inositol accumulation in cultured bovine lens epithelial cells (BLECs) occurred by both high- and low affinity, Nat-dependent transport sites. High ambient glucose significantly inhibited myo-[ 3 H]-inositol uptake; the co-administration of sorbinil, an aldose reductase inhibitor, prevented the inhibitory effect on the low affinity transport site. A glucose-sensitive process for myo-[3 H]-inositol uptake on the high-affinity transport site was uncovered by Lineweaver-Burk analysis. Dixon plot analysis confirmed that the effect of glucose was due to competitive inhibition of the high-affinity myo-inositol transport site while the effect of sorbitol was due to competitive inhibition of the low-affinity myo-inositol transport site.
Date: August 1992
Creator: Chen, Hai-Qing
Partner: UNT Libraries

Pre-Steady State Kinetics of the NAD-Malic Enzyme from Ascaris suum in the Direction of Oxidative Decarboxylation of L-Malate

Description: Stopped-flow experiments in which the NAD-malic enzyme was preincubated with different reactants at near saturating substrate concentrations suggest a slow isomerization of the E:NAD:Mg complex. The lag is eliminated by preincubation with Mg˙² and malate suggesting that the formation of E:Mg:Malate either bypasses or speeds up the slow isomerization step. Circular dichroic spectral studies of the secondary structural changes of the native enzyme in the presence and absence of substrates supports the existence of conformational changes with NAD˙ and malate. Thus, a slow conformational change of the E:NAD:Mg complex is likely one of the rate-limiting steps in the pre-steady state.
Date: December 1991
Creator: Rajapaksa, Ranjani, 1949-
Partner: UNT Libraries

Existence of an Alpha One-Adrenoceptor-Mediated Coronary Vasoconstrictor Reflex During Acute Systemic Hypoxia, in Anesthetized, Open-Chest Dogs

Description: The presence of an alpha-adrenoceptor--mediated coronary vasoconstrictor reflex during acute systemic hypoxia was examined in thirteen chloralose-anesthetized dogs. Local vasodilator effects were avoided by perfusing the left common coronary artery (LCC) with normoxic blood, while the dogs were ventilated with 5% 02-95% N2 . Left ventricular afterload was held constant and positive cardiac inotropic responses and beta two-adrenoceptor-mediated coronary vasodilation were blocked by propranolol. Parasympatheticmediated bradycardia and coronary vasodilation were blocked with atropine. Systemic hypoxia decreased LCC flow to normoxic myocardium by 19.4+2.6 %. Although myocardial oxygen extraction increased 9.7+2.9 %, myocardial oxygen consumption decreased 16.5+2.6 %. Intracoronary prazosin prevented the reflex vasoconstriction during repeated hypoxia.
Date: December 1987
Creator: Grice, Derald Preston
Partner: UNT Libraries

The Determination of Organic-Bound Chlorine Levels in Municipal Wastewaters After Treatment with Heavy Chlorine Doses

Description: The development of an analytical method for the determination of total organic-bound chlorine (TOCl) produced during the chlorination of municipal wastewater effluents is presented. Sewage effluent from the Denton, Texas municipal treatment plant was chlorinated at high chlorine doses (1000 - 4000 ppm), as well as typical treatment levels. Chlororganics present in the wastewater, before and after chlorination, were concentrated by adsorption on Amberlite XAD-2 macroreticular resin, followed by elution with diethyl ether. After concentration, the extracts were analyzed for TOC1 by microcoulometry. Analysis of wastewater extracts revealed the production of substantial amounts of new chlorinated organics when effluents were treated with chlorine. The method shows good precision and estimated accuracy is favorable.
Date: May 1976
Creator: Smith, Garmon B.
Partner: UNT Libraries

The Chlorination of Amino Acid in Municipal Waste Effluents

Description: In model reaction systems to test amino acids in chlorinated waste effluents, several amino acids were chlorinated at high chlorine doses. (2000-4000 mg/1). Amino acids present in municipal waste effluents before and after chlorination were concentrated and purified using cation exchange and Chelex resins. After concentration and cleanup of the samples, the amino acids were derivatized by esterification of the acid functional groups and acylation of the amine groups. Identification and quantification of the amino acids and chlorination products was carried out by gas chromatography/mass spectrometry, using a digital computer data system. Analysis of the waste products revealed the presence of new carbon-chlorine bonded derivatives of the amino acid tyrosine when the effluents were treated with heavy doses of chlorine.
Date: July 1977
Creator: Burleson, Jimmie L.
Partner: UNT Libraries

Isolation and Partial Characterization of Lecithin Cholesterol Acyltransferase and High Density Lipoprotein from Hog Plasma

Description: Lecithin:cholesterol acyltransferase (LCAT) was purified 30,000-fold from hog plasma in a homogeneous state as indicated by polyacrylamide gel electrophoresis. The purified enzyme had an apparent molecular weight of 66,000 and was found to contain about 21.4 percent (w/w) carbohydrate. The properties of hog LCAT including amino acid composition were compared with human LCAT. High density lipoprotein (HDL) was isolated from the hog plasma by an immunoaffinity column chromatography. The isolated HDL showed nearly identical lipid-protein composition although it contained additional protein components when it was compared to HDL isolated by a traditional method involving ultracentrifugation.
Date: May 1984
Creator: Park, Yong Bok
Partner: UNT Libraries

Studies on the Biological Activity of N-nitrosamines

Description: Two aspects of the biological activity of N-nitrosamines were studied. First, the effect of ascorbate on the mutagenicity of N-nitrosopiperidines was studied in the Ames Salmanella/ mammalian microsome mutagenicity test. The addition of ascorbate significantly enhanced the mutagenicity of these compounds. This enhancement was selective for N-nitrosamines suggesting a possible role of ascorbate in N-nitrosamine induced carcinogenicity. Second, the technique of velocity sedimentation in alkaline sucrose density gradients was applied to the detection of N-nitrosamine induced DNA damage in Balb/c 3T3 cells. This technique detected N-nitrosamine induced DNA damage when the cells were made permeable before treatment. This technique compares favorably with other test systems used to evaluate N-nitrosamines and should be useful in further studies of N-nitrosamines.
Date: August 1980
Creator: Barton, Rodney A. (Rodney Alan)
Partner: UNT Libraries

DNA-DNA Hybridization of Methane Oxidizing Bacteria

Description: Bacteria classified in the family Methylomonadaceae must derive their carbon from one-carbon compounds. They are characterized by the possession of internal membranes of two types. Type I membranes are layered and fill the middle of the cells while type II membranes form concentric layers around the periphery of the cells. Also, there are two metabolic pathways by which the methylobacteria assimilate one-carbon compounds. Further evidence of this dichotomy was sought by DNA-DNA saturation hybridization of DNAs from both types of methylobacteria. Very low DNA-DNA homology was seen between types I and II or within the types. It was not possible, therefore, to correlate the degree of genetic relatedness with either the nature of the internal membranes or the pathway of carbon assimilation.
Date: December 1976
Creator: Ackerson, Jill W.
Partner: UNT Libraries

Tolerance to the Behavioral Effects of Methylphenidate

Description: Thirty-one rats were trained on a differential reinforcement of low rate schedule. After responding had stabilized, animals were injected with methylphenidate, twice weekly, presession. Methylphenidate produced dose-dependent increases in response rates and decreases in reinforcements. Repetition of these doses produced a reduced drug effect, and a third administration of the 10 mg/kg dose further reduced the drug effect. Subsequently, the effects of daily and intermittent administration were determined for this dose. Daily methylphenidate, pre-session, produced tolerance to the behavioral effects of methylphenidate and cross-tolerance to the amphetamines. Twice-weekly methylphenidate, pre-session, produced partial tolerance to methylphenidate and partial cross-tolerance to the amphetamines. Thus, periodic exposure to the behaviorally disruptive effects of a drug of the amphetamine class reduces the effects of subsequent exposure.
Date: May 1979
Creator: Brewin, Anne M.
Partner: UNT Libraries

Studies of L-Asparaginase from Lactobacillus Plantarum

Description: This study is concerned with the regulation of Lasparaginase (LA) in the cell-free crude extracts from Lactobacillus plantarum (ATCC8014). A previously reported finding that adenosine triphosphate (ATP) inhibits the action of LA in crude extracts was confirmed. The study was extended to include the mono-, di-, and triphosphates of adenosine, guanosine, cytidine, and uridine. These compounds were also shown to inhibit LA activity. These andother studies revealed that LA appears to be an allosteric type enzyme exhibiting positive homotropism with respect to substrate and heterotropism with respect to the nucleotides tested. The regulation of LA activity by high energy compounds, when coupled with asparagine synthetaseL suggests a relationship between amide synthesis-amide degradation and the energy levels of the cell.
Date: May 1979
Creator: Nalepka, Edward R.
Partner: UNT Libraries

Isolation and Characterization of Proteus vulgaris Methylglyoxal Synthetase

Description: Methylglyoxal synthetase, which catalyzes the formation of methylglyoxal and inorganic phosphate from dihydroxyacetone phosphate, was found in extracts of Proteus vulgaris. An efficient purification procedure utilizing ion exchange column chromatography and isoelectric focusing has been developed. Homogeneity of the enzyme preparation was confirmed by polyacrylamide gel electrophoresis and rechromatography.Two components of methylglyoxal synthetase were obtained upon isoelectric focusing. A comparison of the chemical and physical properties of the two components was carried out. The enzyme is a dimer. In the presence of inorganic phosphate, the hyperbolic saturation kinetics with dihydroxyacetone phosphate are shifted to sigmoidal.
Date: May 1975
Creator: Tsai, Pei-Kuo
Partner: UNT Libraries

Alternate Substrates and Isotope Effects as a Probe of the Malic Enzyme Reaction

Description: Dissociation constants for alternate dirmcleotide substrates and competitive inhibitors suggest that the dinucleotide binding site of the Ascaris suum NAD-malic enzyme is hydrophobic in the vicinity of the nicotinamide ring. Changes in the divalent metal ion activator from Mg^2+ to Mn^2+ or Cd^2+ results in a decrease in the dinucleotide affinity and an increase in the affinity for malate. Primary deuterium and 13-C isotope effects obtained with the different metal ions suggest either a change in the transition state structure for the hydride transfer or decarboxylation steps or both. Deuterium isotope effects are finite whether reactants are maintained at saturating or limiting concentrations with all the metal ions and dinucleotide substrates used. With Cd^2+ as the divalent metal ion, inactivation of the enzyme occurs whether enzyme alone is present or is turning over. Upon inactivation only Cd^2+ ions are bound to the enzyme which becomes denatured. Modification of the enzyme to give an SCN-enzyme decreases the ability of Cd^2+ to cause inactivation. The modified enzyme generally exhibits increases in K_NAD and K_i_metai and decreases in V_max as the metal size increases from Mg^2+ to Mn^2+ or Cd^2+, indicative of crowding in the site. In all cases, affinity for malate greatly decreases, suggesting that malate does not bind optimally to the modified enzyme. For the native enzyme, primary deuterium isotope effects increase with a concomitant decrease in the 13-C effects when NAD is replaced by an alternate dinucleotide substrate different in redox potential. This suggests that when the alternate dinucleotides are used, a switch in the rate limitation of the chemical steps occurs with hydride transfer more rate limiting than decarboxylation. Deuteration of malate decreases the 13-C effect with NAD for the native enzyme, but an increase in 13-C effect is obtained with alternate dinucleotides. These suggest the presence of a ...
Date: August 1988
Creator: Gavva, Sandhya Reddy
Partner: UNT Libraries

Physical, Chemical and Catalytic Properties of the Isozymes of Bovine Glucose Phosphate Isomerase

Description: Glucose phosphate isomerase (GPI) occurs in different bovine tissues as multiple, catalytically active isozymes which can be resolved by polyacrylamide gel electrophoresis and isoelectric focusing. GPI from bovine heart was purified to homogeneity and each of the isozymes was resolved. Four of the five isozymes were characterized with regard to their physical, chemical and catalytic properties in order to establish their possible physiological significance and to ascertain their molecular basis. The isozymes exhibited identical native (118 Kd) and subunit (59 Kd) molecular weights but had different apparent pi values of 7.2, 7.0, 6.8 and 6.6. Structural analyses showed that the amino terminus was blocked and the carboxyl terminal sequence was -Glu-Ala-Ser-Gly for all four isozymes. The most basic isozyme was more stable than the more acidic isozymes (lower pi values) at pH extremes, at high ionic strength, in the presence of denaturants or upon exposure to proteases. Kinetic constants, such as turnover number, Km and Ki values, were identical for all isozymes. Identical amino acid composition and peptide mapping by chemical cleavage at methionine and cysteine residues of the isozymes suggest a postsynthetic modification rather then a genetic origin for the in vivo isozymes. When the most basic isozyme was incubated in vitro under mild alkaline conditions, there was a spontaneous generation of the more acidic isozymes with electrophoretic properties identical to those found in vivo. The simultaneous release in ammonia along with the spontaneous shift to more acidic isozymes and changes in the specific cleavage of the Asn-Gly bonds by hydroxylamine of the acidic isozyme indicates deamidation as the probable molecular basis. In summary the isozymes appear to be the result of spontaneous, postsynthetic modifications involving the addition of an equal number of negative charges and are consistent with the deamidation process.
Date: August 1987
Creator: Cini, John Kenneth
Partner: UNT Libraries

Regulation of Lactobacillic Acid Formation in Lactobacillus Plantarum

Description: Cyclopropanation of the unsaturated fatty acid moieties of membrane phospholipids is a commonly observed phenomenon in a number of bacterial systems. The cyclopropane fatty acids are usually synthesized during and after the transition from exponential growth to stationary phase, or under such environmental conditions as acidic culture pH, low oxygen tension or high salt concentrations. S-Adenosylmethionine, the ubiquitous methyl group donor, provides the methylene bridge carbon in the reaction catalyzed by cyclopropane fatty acid synthase. Also formed in the reaction is S-adenosylhomocysteine, a potent inhibitor of cyclopropane fatty acid synthase, which is degraded by S-adenosylhomocysteine nucleosidase. This work provides evidence for at least two modes of regulation of lactobacillic acid synthesis, the cyclopropane fatty acid formed from cis-vaccenic acid (cis-11,12-octadecenoic acid), in Lactobacillus piantarum.
Date: December 1980
Creator: Smith, Darwin Dennis
Partner: UNT Libraries

Synthesis of Anthracyclines Related to Adriamycin

Description: This dissertation reports the preparation of several types of anthraquinones structurally related to adriamycin. It describes the synthesis of two types of 2-aminoquinizarin compounds. It also presents two new syntheses of a heterocyclic tetracyclic ring system, similar to the aglicone ring system of adriamycin. A series of 2-aminoquinizarins was prepared by adding several primary amines to quinizarin. Quinizarin was shown to be essentially inert toward secondary amines. Several secondary amine adducts with quinizarin have been prepared, however, by treating the bis-boroacetate ester of quinizarin with the amines. Both types of 2-aminoquinizarin compounds exhibit outstanding potential for possessing antineoplastic activity, and several have been submitted to the National Cancer Institute for testing in their screening program for antineoplastic agents.
Date: May 1981
Creator: White, Roger J.
Partner: UNT Libraries

Factors Inhibiting Dissociation Of Neisseria gonorrhoeae Cells

Description: The initial studies reported in this dissertation were attempts to induce mutations in those genes which control dissociation in cells of Nei sseria gonorrhoeae. These studies led to an investigation of survival curves of cells grown in liquid media. Instead of survival curves reflecting the diploid nature of gonococci, multiple cell kinetics were observed. It was found that large clumps contained a predominance of cells of the T2 type and that when these clumps were dispersed by DNAase, it appeared that dissociation of T2 was inhibited. The notion of a mechanism of T2 to T4 dissociation being due to genetic transformation was disspelled by these data.
Date: August 1982
Creator: Gonzalez, Anthony H.
Partner: UNT Libraries

Purification and Studies of Mammalian Glyoxalase Enzymes

Description: The glyoxalase system, which has been known since 1913, is widely distributed in nature. The system consists of two enzymes, glyoxalase I and glyoxalase II. Methylglyoxal is very unstable and undergoes oxidation and polymerization reactions. One of the purposes of this study was to find a simple, convenient and reproducible method of methylglyoxal preparation. Another objective was the purification of both glyoxalase enzymes employing affinity chromatography as a major step. The purified enzymes were to be characterized by chemical, physical and kinetic properties as an approach to the understanding of the biological function of the system.
Date: December 1980
Creator: Oray, Bedii
Partner: UNT Libraries

Poly(ADP-ribose) Synthesis as a Function of Growth and DNA Fragmentation

Description: This work examines the synthesis of poly(ADP-ribose) in normal and SV40-transformed monolayer cultures of 3T3 cells as a function of growth and DNA fragmentation. A review of the relevant literature is given in the introduction of this work. Poly(ADP-ribose) synthesis has been implicated in transcription, replication, repair, differentiation and regulation of cell growth. The results of this study suggest that poly(ADP-ribose) synthesis is involved in some aspect of cell-growth control and DNA repair.
Date: December 1981
Creator: Levi, Viktorya
Partner: UNT Libraries

Quantification of Poly(ADP-ribose) in Normal and in DNA-Damaged Cells

Description: This work presents the development of a new highly sensitive and selective chemical assay for poly(ADP-ribose) which is routinely useful for the determination of polymer levels in vivo. This method was used to carefully measure poly(ADP-ribose) levels in normal and in DNA-damaged cells. The results of these studies strongly suggest that synthesis of poly(ADP-ribose) is involved in some aspect of DNA repair. A review of the literature is presented in the introduction of this work. Poly(ADP-ribose) synthesis has been implicated in aspects of transcription, in DNA syn thesis, and in DNA repair largely based on evidence from in vitro studies. It is apparent that current methodology has not allowed the routine quantification of poly(ADP-ribose) in vivo, hence the lack of i^n vivo data concerning the function(s) of the polymer. The body of this work presents the development of two chemical methods for the quantification of poly(ADP-ribose) and the application of one of these methods to the measurement of polymer levels in normal and DNA-damaged cells. Preliminary studies are presented on the utilization of combined gas chromatography/mass spectroscopy for the selective quantification of nucleoside derivatives. A second method makes use of the unique chemistry of the polymer for quantification. The polymer was selectively adsorbed to dihydroxyboryl-sepharose which allowed the removal of most RNA, DNA, and protein from the samples. The polymer was hydrolyzed to the unique nucleoside 2'—^-l*'-ribosyladenosine by digestion with venom phosphodiesterase and bacterial alkaline phosphatase. The 1-N^-etheno derivative of ribosyladenosine was formed by reaction with chloroacetaldehyde and this derivative was seperated from other fluorescent species by reversed phase high pressure liquid chromatography.
Date: December 1980
Creator: Sims, James L.
Partner: UNT Libraries